Abstract Histidine-lysine (HK) peptides can be tailored to transport different forms of nucleic acids such as plasmids or siRNA into cells by altering their degree of branching and amino acid sequence. The HK carrier has shown value in treating cancer, hypertrophic scars, and liver fibrosis in pre-clinical models. In contrast to the effective in vitro highly-branched H2K4b peptide carrier of plasmids, the linear H2K peptide is a poor carrier in vitro, yet surprisingly, H2K nanoplexes target tumors with very high transfection efficiency. Because H2K4b and H2K plasmid nanoplexes differ in their accumulation and distribution within the tumor, there are clearly other mechanisms besides EPR that govern the tumor specificity of H2K nanoplexes. Because H2K has a repeating sequence pattern of ?KHHK-, transcytosis of the nanoplex mediated by the neuropilin-1 receptor (NRP1) through the tumor endothelium provides a rationale for enhanced tumor targeting and accumulation. Although additional development concerning the linear peptide is required, these early findings are encouraging and are the focus of this application. To test the overall hypothesis that characterization of HK nanoplexes containing a shRNA plasmid targeting Kras/Raf-1 will facilitate improved antitumor activity, the following aims are planned. Aim 1 will determine the structure-activity relationship of HK peptides as carriers of plasmids in vivo. To transport the shRNA-inhibitory plasmids systemically to tumors, we have selected several HK polymers with a predominant repeating amino acid sequence of ?KHHK- (i.e., H2K), an effective sequence for plasmid transport in vivo. By combining the H2K sequence with other amino acid sequence patterns, and varying the length and number of branches, we hypothesize that derivatives of H2K will be identified that are more effective in vivo carriers of plasmids targeting different breast tumor models. Aim 2 will delineate the biophysical and biological properties of HK nanoplexes essential for transfection. Several biophysical techniques will be examined, including morphology, binding, zeta potential, and stability of HK plasmid nanoplexes to correlate their physical properties with the efficacy of plasmid delivery to tumors. By comparing HK peptides that differ in their branching and length with in vitro and in vivo biophysical methods, our hypothesis is that key structural features of the carrier will be identified that will affect stability and distribution within the tumor and increase gene expression. Based on these structural and mechanistic studies, Aim 3 will develop a targeted RNAi therapeutic with an optimized HK nanoplex toward breast tumors in mice. We hypothesize that the specific patterns based on the linear H2K peptide and elevated levels of NRP1 in tumors will markedly augment delivery of tumor-inhibitory plasmids (shRaf-1 and shKras) to orthotopic or metastatic tumors. Moreover, addition of a tumor-targeting ligand to the HK nanoplex should increase the accumulation and specificity of the nanoplex to the tumor. These results of improved delivery to the tumor should translate to other pathological disease such as liver fibrosis/cirrhosis, which also have elevated levels of NRP-1.